Disadvantages of the antibiotic selectable marker system. Most transformation techniques co-introduce a gene that confers antibiotic resistance, along with the gene of interest to impart a desired trait. Regenerating transformed cells in antibiotic containing growth media permits selection of only those cells that have incorporated the foreign genes as the gene of interest. Once transgenic plants are regenerated, antibiotic resistance genes serve no useful purpose but they continue to produce their gene products. One of the primary concerns of genetically modified (GM) crops is the presence of clinically important antibiotic resistance gene products in transgenic plants that could inactivate oral doses of the antibiotic (reviewed by Puchta 2000; Daniell 1999A). Another concern is that the antibiotic resistant genes could be transferred to pathogenic microbes in the gastrointestinal tract or soil rendering them resistant to treatment with such antibiotics. Antibiotic resistant bacteria are one of the major challenges of modern medicine. In Germany, GM crops containing antibiotic resistant genes have been banned from release (Peerenboom 2000).
Plastid genetic engineering as an alternative to nuclear genetic engineering. Plastid genetic engineering, particularly chloroplast genetic engineering, is emerging as an alternative new technology to overcome some of the environmental concerns of nuclear genetic engineering (reviewed by Bogorad, 2000). One common environmental concern is the escape of foreign gene through pollen or seed dispersal from transgenic crop plants to their weedy relatives creating super weeds or causing genetic pollution among other crops (Daniell 1999B). Keeler et al. (1996) have summarized valuable data on the weedy wild relatives of sixty important crop plants and potential hybridization between crops and wild relatives. Among sixty crops, only eleven do not have congeners and the rest of the crops have wild relatives somewhere in the world. In addition, genetic pollution among crops has resulted in several lawsuits and shrunk the European market of Canadian organic farmers (Hoyle 1999). Several major food corporations have required segregation of native crops from those “polluted” with transgenes. Two legislations have been submitted in the U.S. to protect organic farmers whose crops inadvertently contain transgenes via pollen drift (Fox 2000). Maternal inheritance of foreign genes through chloroplast genetic engineering is highly desirable in such instances where there is potential for out-cross among crops or between crops and weeds (Daniell et al. 1998; Scott and Wilkinson 1999; Daniell 1999C).
Yet another concern in the use of nuclear transgenic crops expressing the Bacillus thuringiensis (Bt) toxins is the sub-optimal production of toxins resulting in increased risk of pests developing Bt resistance. Plant-specific recommendations to reduce Bt resistance development include increasing Bt expression levels (high dose strategy), expressing multiple toxins (gene pyramiding), or expressing the protein only in tissues highly sensitive to damage (tissue specific expression). All three approaches are attainable through chloroplast transformation (Daniell 1999C). For example, hyperexpression of several thousand copies of a novel B.t. gene via chloroplast genetic engineering, resulted in 100% mortality of insects that are up to 40.000-fold resistant to other B.t. proteins (Kota et al. 1999). Another hotly debated environmental concern expressed recently is the toxicity of transgenic pollen to non-target insects, such as the Monarch butterflies (Losey et al. 1999; Hodgson 1999). Although pollen from a few plants shown to exhibit maternal plastid inheritance contains metabolically active plastids, the plastid DNA itself is lost during the process of pollen maturation and hence is not transmitted to the next generation (reviewed in Heifetz, 2000, Bock and Hagmann, 2000). Lack of insecticidal protein in transgenic pollen engineered via the chloroplast genome with the cry2A gene has been demonstrated recently, even though chloroplast in leaves contained as much as 47% CRY protein of the total soluble protein (De Cosa et al. 2000).
The need for alternatives to the antibiotic selectable marker system. Despite these advantages, one major disadvantage with chloroplast: genetic engineering in higher plants may be the utilization of the antibiotic resistance genes as the selectable marker to confer streptomycin/spectinomycin resistance. Initially, selection for chloroplast transformation utilized a cloned mutant 16S rRNA gene that does not bind the antibiotic and this conferred spectinomycin resistance (Svab et al. 1990). Subsequently, the aadA gene product that inactivates the antibiotic by transferring the adenyl moiety of ATP to spectinomycin/streptomycin was used (Svab and Maliga 1993). These antibiotics are commonly used to control bacterial infection in humans and animals. The probability of gene transfer from plants to bacteria living in the gastrointestinal tract or soil may be enhanced by the compatible protein synthetic machinery between chloroplasts and bacteria, in addition to presence of thousands of copies of the antibiotic resistance genes per cell. Also, most antibiotic resistance genes used in genetic engineering originate from bacteria.
Because of the presence of thousands of antibiotic resistant genes in each cell of chloroplast transgenic plants and the use of the most commonly used antibiotics in the selection process, it is important to develop a chloroplast genetic engineering approach without the use of antibiotics.
Non-obviousness of antibiotic free selection. Despite several advantages of plastid transformation, one major disadvantage with chloroplast genetic engineering in higher plants is the utilization of the antibiotic resistance genes as the selectable marker. Initially, selection for chloroplast transformation utilized a cloned mutant 16S rRNA gene that did not bind the antibiotic and this conferred spectinomycin resistance. Subsequently, the aadA gene was used as a selectable marker. Aminoglycoside 3′-adenylyltransferase inactivates the antibiotic by transferring the adenyl moiety of ATP to spectinomycin/streptomycin. Unfortunately, bacterial infections in humans and animals are also controlled by using these antibiotics. The probability of gene transfer from plants to bacteria living in the soil or gastrointestinal tract may be enhanced by the compatible protein synthetic machinery between chloroplasts and bacteria, in addition to presence of thousands of copies of the antibiotic resistance genes per cell. Also, most antibiotic resistance genes used in genetic engineering originate from bacteria.
Prior to this invention, there was no report of modifying the plastid genome without the use of antibiotic selection. Daniell et al. (2001) reported the first genetic engineering of the higher plant chloroplast genome without the use of antibiotic selection. The betaine aldehyde dehydrogenase (BADH) gene from spinach was used as a selectable marker. The selection process involves conversion of toxic betaine aldehyde (BA) by the BADH enzyme to nontoxic glycine betaine, which also serves as an osmoprotectant. While it was known earlier that BADH was a plant enzyme, it could not be conclusively demonstrated that this was a chloroplast enzyme because it lacked the typical transit peptide found in all chloroplast proteins imported from the cytosol.
The absence of a typical transit peptide raised several questions about proper cleavage of BADH enzyme in the stroma within plastids to be fully functional. It was not known whether the BADH enzyme would be catalytically active without proper cleavage within plastids.
The nuclear BADH cDNA with high GC content was never anticipated to express well in the AT rich prokaryotic plastid compartment because the codon usage is very different between the prokaryotic chloroplast compartment and the eukaryotic nuclear compartment. Therefore, it was not obvious to express a nuclear gene in the plastid compartment.
When the chloroplast transformation system was developed, it was hypothesized that the transformation process is possible only under non-lethal selection. Accumulation of betaine aldehyde is toxic and lethal to plant cells. Therefore, it was not clear whether non-lethal selection was required for chloroplast transformation. This invention has confirmed that the only requirement was that the selection process should be specific to plastids, particularly chloroplasts.
Rapid regeneration of chloroplast transgenic plants obtained under BA selection was never anticipated or suggested in any prior art. Chloroplast transformation efficiency was 25 fold higher in BA selection than spectinomycin and this was never anticipated in any previous investigations. Higher efficiency of betaine aldehyde selection compared to spectinomycin should facilitate chloroplast transformation of many economically important crops, including cereals that are naturally resistant to spectinomycin, in addition to conferring salt/drought tolerance.
Use of genes that are naturally present in spinach for selection, in addition to gene containment, should ease public concerns regarding GM crops.